Given the potential catastrophic effects of inappropriate activation of signaling cascades, it is essential that the gene targets of signaling pathways are maintained in a repressed state in the absence of activating ligand. It is assumed that packaging of DNA into nucleosomes, and positioning of nucleosomes over gene regulatory elements can block transcription. Members of the ISWI family of ATP-dependent chromatin remodeling enzymes are key regulators of nucleosome positioning and, this report has shown that NURF activity is required to maintain repression of JAK/STAT target genes. Repression by NURF is consistent with other studies of ISWI chromatin remodeling enzymes. For example, the yeast Isw2 remodeling complex is required for transcriptional repression. In humans, the Snf2h-containing chromatin remodeling complex NoRC slides nucleosomes to silence rRNA genes. More recently, ISWI in African trypanosomes has been demonstrated to silence variant surface glycoprotein gene expression sites (Kwon, 2008 and references therein).

Although a chromatin remodeling enzyme (SWI/SNF) is required for activation of STAT-inducible genes, this is the first report to implicate a chromatin remodeling enzyme in repression of JAK/STAT target genes. There is, however, evidence that covalent histone modification is involved in repression of JAK/STAT target genes. The co-repressor SMRT suppresses induction of STAT5 target genes. This suppression is blocked by the addition of the histone deacetylase inhibitor TSA, implying a chromatin component in repression. In Drosophila, mutations in the heterochromatin component HP1 have been shown to enhance tumor formation in hopTum gain-of-function JAK mutants, further implying a connection between chromatin, JAK/STAT and transcriptional repression (Kwon, 2008).

It cannot be excluded that some of the genes that show increases in expression in the Nurf301 and hopTum mutants may be indirect targets of NURF. Changes in the proportion of lamellocytes in these mutant backgrounds may affect transcription of some genes, for example the Drosophila β-integrin subunit mys. Nevertheless by ChIP it has been shown that NURF is located at the promoters of two potential targets, CG5791 and dei. Importantly, NURF-biding coincides with recognition sequences for STAT are overlapped by binding sites for the transcriptional repressor Ken. In addition, it was shown that NURF physically interacts with Ken, providing a means by which NURF can be recruited to JAK/STAT target genes (Kwon, 2008).

The data suggests a mechanism by which Ken represses transcription. It is proposed that in unstimulated conditions Ken binds to JAK/STAT target promoters and recruits NURF. NURF-mediated nucleosome-sliding then establishes a repressed chromatin configuration that blocks transcription, perhaps by positioning a nucleosome over the transcription start site. Upon stimulation Stat92E enters the nucleus, binds target promoters and, in addition to recruiting co-activators, displaces Ken and thus NURF. The promoter is switched from a repressive to active chromatin state, and transcription can occur. In NURF mutants, it is suggested that repressive nucleosome positions are either not established or maintained and, consequently, JAK/STAT targets are not silenced. As a result, transcription can occur in the absence of JAK/STAT activation (Kwon, 2008).

More than two decades ago, Travers and colleagues proposed that underlying DNA sequence can influence nucleosome positioning, with some sequences favoring, and others destabilizing nucleosomes. Recent computational analysis has revealed that sequences at yeast transcription start-sites encode nucleosomes that are intrinsically unstable. The NURF-related yeast Isw2 chromatin remodeling complex is able to override these refractory sequences, positioning nucleosomes over them, to block promoters. Interestingly, in ISW2 mutants, nucleosomes revert to thermodynamically favorable positions exposing the promoter. It is speculated that Drosophila transcription start-sites may similarly be refractory to nucleosomes. Normally, at JAK/STAT targets, NURF overrides these sequences but, in NURF mutants, these transcription start sites may similarly be exposed (Kwon, 2008 and references therein).

In the case of the innate immune system, prompt activation of signaling cascades such as the JAK/STAT pathway in response to pathogens are essential for survival. However, it is also paramount that in the absence of challenge the innate immune system be held in check or regulated, to prevent inappropriate damage. In humans chronic immune-mediated inflammatory conditions are characterized by the abnormal or continued episodic activation of these pathways leading to disease. Drosophila NURF has a vital function in preventing ectopic activation of the JAK/STAT pathway. In the absence of NURF, Drosophila develop an immune-mediated inflammatory syndrome -- melanotic tumors. Given the conservation of NURF between Drosophila and humans, it is tempting to speculate that human NURF may function to hold inflammatory pathways in check (Kwon, 2008).

Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the JAK-STAT pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somatic cyst stem cells (CySCs). This study found that ken and barbie (ken) is autonomously required for the self-renewal of CySCs but not GSCs. Furthermore, Ken misexpression in the CySC lineage induces the cell-autonomous self-renewal of somatic cells as well as the nonautonomous self-renewal of germ cells outside the niche. Thus, Ken, like Stat92E and its targets ZFH1 and Chinmo, is necessary and sufficient for CySC renewal. However, ken is not a JAK-STAT target in the testis, but instead acts in parallel to Stat92E to ensure CySC self-renewal. Ken represses a subset of Stat92E targets in the embryo suggesting that Ken maintains CySCs by repressing differentiation factors. In support of this hypothesis, it was found that the global JAK-STAT inhibitor Protein tyrosine phosphatase 61F (Ptp61F) is a JAK-STAT target in the testis that is repressed by Ken. Together, this work demonstrates that Ken has an important role in the in the inhibition of CySC differentiation. Studies of ken may inform understanding of its vertebrate orthologue B-Cell Lymphoma 6 (BCL6) and how misregulation of this oncogene leads to human lymphomas (Issigonis, 2012).

This study demonstrates that ken, the orthologue of the human oncogene BCL6, plays a novel and crucial role in adult stem cell maintenance. Furthermore, the data show that ken is sufficient to promote the self-renewal of CySCs outside of their normal niche, which in turn drives the nonautonomous self-renewal of GSCs. This is consistent with previous studies, which have shown that hyperactivation of JAK-STAT signaling or misexpression of the Stat92E targets ZFH1 or Chinmo are sufficient to induce ectopic CySCs and GSCs. This work also reveals a previously unappreciated role for Stat92E in the Drosophila testis- transcriptional repression of target genes (Issigonis, 2012).

This study demonstrates the importance of ken in maintaining CySC fate. The only three genes other than Stat92E currently known to be necessary and sufficient for CySC self-renewal are ken, zfh1

, and chinmo. Remarkably, all three genes are known to behave as transcriptional repressors. Furthermore, both ken and chinmo encode proteins that share the same overall domain structure: an N-terminal BTB domain and C-terminal DNA-binding zinc fingers. The Drosophila genome encodes 32 BTB-ZF proteins, so it would be interesting to see whether other BTB-ZF proteins are also sufficient to induce ectopic CySCs and GSCs when expressed in the CySC lineage. BTB-ZF proteins regulate many important biological processes such as cell survival and differentiation and generally behave as transcriptional repressors. Therefore, it is clear that transcriptional repression plays a critical role in regulating CySC fate (Issigonis, 2012).

It will be interesting to learn whether Ken, ZFH1, and Chinmo each control a distinct set of genes, or whether some of their targets are co-regulated. Both ZFH1 and BCL6, the mammalian homolog of Ken, are known to interact with the corepressor CtBP (C-terminal binding protein). Furthermore, heterodimerization between different BTB-ZF family members has been shown to occur. Since the transcriptional repressors Ken, ZFH1, and Chinmo have similar loss-of-function phenotypes (CySC loss) and gain-of-function phenotypes (ectopic CySCs), it seems likely that identifying their common targets will lead to identification of key effectors required to promote CySC self-renewal. An important parallel can be drawn to studies on embryonic stem (ES) cells which demonstrate that the main ES cell self-renewal factors OCT4, SOX2, and NANOG promote stem cell fate by transcriptionally repressing genes required for differentiation. Interestingly, OCT4, SOX2, and NANOG have been shown to co-occupy a number of target genes. Mapping Ken as well as ZFH1 and Chinmo to their binding sites within CySCs will reveal how these transcriptional regulators behave to promote self-renewal and block differentiation (Issigonis, 2012).

Previous studies have uncovered the dependence of the germ cells on CySCs for their self-renewal and on cyst cells for their proper differentiation. However, further investigation is required to elucidate the mechanisms by which ectopic CySCs are induced, and how this consequently leads to GSC self-renewal. It is unknown whether blocking differentiation in CySCs is sufficient to stall GSCs in an undifferentiated state or whether CySCs send a signal to neighboring germ cells causing them to self-renew. This work and previous studies have begun to uncover the regulatory network comprised of transcription factors and chromatin remodelers (Cherry, 2010) in CySCs. In order to understand how these transcriptional regulatory networks control the decision between stem cell fate versus differentiation in CySCs, and how CySC self-renewal promotes GSC identity, one must identify the downstream target genes of these critical transcriptional regulators (Issigonis, 2012).

Previous work from several labs has shown the importance of JAK-STAT activity for the maintenance of both CySCs and GSCs. In CySCs, JAK-STAT signaling promotes stem cell identity by activating the transcription of self-renewal factors, and in GSCs, pathway activation primarily regulates their adhesion to the hub. However, attenuation of JAK-STAT signaling is critical as well; expression of the Stat92E target Socs36E in CySCs is necessary to create a negative feedback loop that prevents CySCs from activating Stat92E at aberrantly high levels and consequently outcompeting neighboring GSCs (Issigonis, 2009). Therefore, differentially fine-tuning the overall global levels of JAK-STAT pathway activation in the two stem cell types is essential. But how do the stem cells precisely regulate which JAK-STAT targets are activated in the appropriate cell lineage? For example, even though the JAK-STAT pathway is activated in both CySCs and GSCs (albeit at different levels), the target genes zfh1 and Socs36E are expressed in the CySCs but not the GSCs. It is possible that distinct STAT targets respond to different thresholds of STAT activation. Furthermore, certain co-activators or co-repressors may be uniquely expressed or may function exclusively in one cell lineage and not the other. For example, ZFH1 is only expressed in CySCs and is required for their maintenance. On the other hand, Chinmo is expressed in both GSCs and CySCs, but functions solely in the latter stem cell population for their maintenance. Ken is enriched in the testis apex, and similar to the transcriptional repressorsZFH1 and Chinmo, is required in CySCs, but not GSCs. However, in the testis, ken is not a target of the JAK-STAT pathway, unlike zfh1 and chinmo. It is worth noting that although their loss-of-function phenotypes are similar, ken mutant CySC clones are lost more slowly than stat92E, zfh1, or chinmo mutant CySCs. One reason for this difference may be attributed to the fact that the available ken alleles are not null. However, it is also possible that genes such as zfh1 and chinmo may have stronger loss-of-function phenotypes because they play a primary role in CySC maintenance whereas Ken may perform secondary functions such as fine-tuning the transcriptional output of the JAK-STAT pathway. The Drosophila testis niche presents a unique opportunity to study how a single signaling pathway regulates two different stem cell populations within a niche via (1) differential regulation of global antagonists (i.e. Socs36E), (2) activation of a distinct set of target genes exclusively in one stem cell type (i.e. zfh1), and (3) differential regulation by transcriptional repressors (i.e. ken and chinmo) (Issigonis, 2012).

An interesting discovery from this study is that Stat92E represses the expression of Ptp61F. STATs were originally discovered as activators of gene transcription in response to interferons. Recently, however, increasing evidence indicates that in addition to their more familiar and well-documented role as transcriptional activators, STATs can also behave as functional repressors in an indirect manner (via STAT-induced activation of a repressor) or directly (through interactions with DNA methyltransferases, histone deacetylases, or heterochromatin proteins) (Issigonis, 2012).

In Drosophila, JAK-STAT pathway activation is known to upregulate the transcription of some targets, while repressing others. However, how a transcription factor such as Stat92E can stimulate the expression of individual genes while inhibiting others that have potentially conflicting roles is not well understood. The Drosophila testis provides a good model system to study this problem; Stat92E is required for the self-renewal of CySCs, presumably by positively regulating genes required for stem-cell identity while repressing those which would lead to opposite fates (i.e. differentiation). The current results indicate that Ptp61F is negatively regulated by JAK-STAT signaling in the testis since the activation of JAK-STAT leads to a dramatic decrease in Ptp61F expression. Since Ptp61F expression was quickly downregulated in hs-upd testes after a single heat-shock pulse, it is thought that Stat92E may be directly repressing Ptp61F transcription instead of activating the expression of a Ptp61F repressor. Support for this comes from work performed in an ex vivo system using Drosophila haemocyte-like cells to identify JAK-STAT targets. Upd or HopTumL stimulation of these haemocyte-like cells leads to a significant increase in the transcript levels of the 'immediate-early' JAK-STAT target Socs36E, which responds within two hours of pathway activation. These observations were recapitulated in vivo, since a robust increase in Socs36E expression levels was observed in response to a heat-shocking protocol in hs-upd testes. Similarly, the rapid response seen in Ptp61F expression levels upon JAK-STAT pathway activation may reflect a direct repression of this target as opposed to a secondary effect. Future studies will address the mechanism by which Stat92E represses the JAK-STAT inhibitor Ptp61F to promote CySC self-renewal (Issigonis, 2012).

While the mechanism by which Ken represses JAK-STAT targets is currently unknown, clues to how Ken may be behaving can be drawn from its orthologue BCL6, which interacts with chromatin modifiers such as SMRT, mSIN3A, N-CoR, BcoR, and histone deacetylases (HDACs). This suggests that Ken may be acting through these partners to block transcriptional activation through chromatin modification. Another possibility is that Ken directly blocks Stat92E from binding to and transcriptionally activating expression of target genes. Furthermore, since Stat92E can either activate or repress expression of targets, it is also possible that Ken behaves as a Stat92E co-repressor. Any of these non-exclusive possibilities will further understanding of how a signaling pathway is able to transcriptionally activate different target genes in different cell types and stages of development as opposed to eliciting the indiscriminate activation of all possible target genes at once (Issigonis, 2012).

Chromosomal rearrangements and point mutations that lead to the misregulation of BCL6 occur frequently in human lymphomas. Furthermore, constitutive overexpression of BCL6 in mice promotes the development of lymphomas. BCL6 has been shown to repress differentiation of B-cells and mammary cells. This study has found that Ken plays an analogous role in repressing differentiation of CySCs in the Drosophila testis. Future studies on Drosophila Ken and its targets will further understanding of the mammalian oncogene BCL6 (Issigonis, 2012).

A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. A new negative regulator of JAK/STAT signaling is described and a potential mechanism identified by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, Ken & Barbie (Ken), which is an ortholog of the mammalian proto-oncogene BCL6, has been identified as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, endogenous Ken is shown specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo (Arbouzova, 2006).

Analysis of phenotypes associated with mutations in Drosophila JAK/STAT pathway components have identified a wide variety of requirements for the pathway during embryonic development and in adults. What is less clear is how the repeated stimulation of a single pathway is able to generate this pleiotropy of developmental functions. In order to identify modulators of JAK/STAT signaling that may be involved in this process, a genetic screen was undertaken for modifiers of the dominant phenotype caused by the ectopic expression of the pathway ligand Unpaired (Upd) in the developing eye imaginal disc. Such misexpression by GMR-updΔ3′ results in overgrowth of the adult eye, a phenotype sensitive to the strength of pathway signaling activity. With this assay, one genomic region, defined by Df(2R)Chig320, was found to enhance the GMR-updΔ3′-induced eye overgrowth phenotype. Of the genes deleted by Df(2R)Chig320, only mutations in ken showed consistent and reproducible enhancement of the phenotype. In addition, other dominant phenotypes induced by transgene expression from the GMR promoter are not modulated by ken mutations, indicating that Ken is unlikely to interact with the misexpression construct used (Arbouzova, 2006).

The enhancement of the GMR-updΔ3′ phenotype after removal of one copy of ken implies that Ken normally functions antagonistically to JAK/STAT signaling. Therefore phenotypes associated with mutations in other pathway components were tested to establish the reliability of this initial observation. Consistent with this, genetic interaction assays between ken mutations and the hypomorphic loss-of-function allele stat92EHJ show a reduction in the frequency of wing vein defects normally associated with this stat92E allele. Moreover, the degree of suppression is consistent with the strength of ken alleles tested. Similarly, the frequency of 'strong' posterior spiracle phenotypes caused by the dome367 allele of the pathway receptor is also reduced when crossed to ken alleles or the Df(2R)Chig320 deficiency, with a concomitant increase in 'weak' phenotypes (Arbouzova, 2006).

Thus, multiple independent ken alleles all modify diverse phenotypes caused by both gain- and loss-of-function mutations in multiple JAK/STAT pathway components. Each of these components acts at different levels of the signaling cascade and show interactions indicating that Ken consistently acts as an antagonist of the pathway (Arbouzova, 2006).

The ken locus contains three exons encoding a 601 aa protein. Ken possesses an N-terminal BTB/POZ domain between aa 17 and 131 and three C-terminal C2H2 zinc finger motifs from aa 502 to 590. Strikingly, a number of Zn finger-containing proteins that also contain BTB/POZ domains have also been shown to function as transcriptional repressors -- often via the recruitment of corepressors such as SMRT, mSIN3A, N-CoR, and HDAC-1 (Arbouzova, 2006).

Searches for proteins similar to Ken identified homologs in Drosophila pseudoobscura and the mosquito Anopheles gambiae. In vertebrates, human B-Cell Lymphoma 6 (BCL6) was the closest full-length homolog. Drosophila Ken and human BCL6 share the same domain structure and show 20.3% overall identity. Proteins listed as potential vertebrate homologs of Ken in Flybase are more distantly related (Arbouzova, 2006).

Expression of ken was also examined during development, where it is detected in a dynamic pattern from newly laid eggs, throughout embryogenesis, and in imaginal discs. As such, endogenous Ken is present in all tissues and stages in which genetic interactions were observed (Arbouzova, 2006).

Given the presence of potentially DNA binding Zn finger domains and the nuclear localization of GFPKen, the DNA binding properties of Ken was determined by using an in vitro selection technique termed SELEX (systematic evolution of ligands by exponential enrichment). With a GST-tagged Ken Zn finger domain and a randomized oligonucleotide library, ten successive rounds of selection were undertaken. Sequencing of the resulting oligonucleotide pool and alignment of 43 independent clones showed that all recovered plasmids were unique and each contained one, or occasionally two, copies of the motif GNGAAAK (K = G/T) (Arbouzova, 2006).

To confirm the SELEX results, GFPKen was expressed in tissue culture cells and these were used for electromobility shift assays (EMSA). A radioactively labeled probe containing the wild-type (wt) consensus binding site GAGAAAG gives a specific band, which can be supershifted by an anti-GFP antibody and therefore represents a GFPKen/DNA complex. In order to identify positions essential for binding, a competition assay was used in which unlabeled oligonucleotides containing single substitutions in each position from 1 to 7 were added to binding reactions. 10-fold excess of unlabeled wild-type consensus oligonucleotide greatly diminished the intensity of the GFPKen band, while 50- and 100-fold excess totally blocked the original signal. By contrast, competition with unlabeled m3 oligonucleotides containing a G to A substitution at position 3 failed to significantly reduce the intensity of the band even at 100-fold excess. With this approach, the positions 1 and 7 are found dispensable for DNA binding, whereas the central GAAA core is absolutely required. Similar results were obtained with the converse experiment with labeled mutant probes, although in this case the wt probe produces a stronger signal than the m1 and m7 mutant oligonucleotides. Taken together, these experiments not only define the core sequence for Ken binding, but also demonstrate the specificity of Ken as a site-specific DNA binding molecule. Interestingly, the core consensus bound by Ken is very similar to that identified for human BCL6, with the Zn fingers of the latter binding to a DNA sequence containing a core GAAAG motif
(Arbouzova, 2006).

One initial observation made is that the core GAAA essential for Ken binding overlaps the sequence recognized by STAT92E. Consistent with this overlap, a 100-fold excess of unlabeled oligonucleotide containing the STAT92E consensus is sufficient to fully compete for Ken in EMSA assays. Given this finding, it is hypothesized that the negative regulation of JAK/STAT signaling by Ken observed in genetic interaction assays may occur via a mechanism of competitive DNA binding site occupation. Due to the incomplete overlap between the STAT92E and Ken core sequences, this hypothesis also implies the existence of STAT92E DNA binding sites to which both STAT92E and Ken could bind (STAT+/Ken+) as well as sites with which Ken cannot associate (STAT+/Ken−) (Arbouzova, 2006).

To test this hypothesis, a cell culture-based assay was set up by using a luciferase-expressing reporter containing four STAT92E binding sites originally identified in the promoter of the Draf locus. In addition to this STAT+/Ken+ wild-type reporter, STAT+/Ken− and STAT−/Ken− variants identical but for the binding sequences were generated. When transfected into the hemocyte-like Kc167 Drosophila cell line, both STAT+/Ken+ and STAT+/Ken− reporters showed strong stimulation upon coexpression with the pathway ligand Upd, an assay previously shown to require an intact JAK/STAT cascade. When cotransfected with KenGFP, the activity of the STAT+/Ken+ reporter was reduced, an effect reproduced in three independent experiments with both KenGFP and Ken. While the reduction in reporter activity for the STAT+/Ken+ assay shown is statistically significant, the STAT+/Ken− reporter was unaffected by the coexpression of Ken. Reporters containing binding sites mutated to prevent binding of both STAT92E and Ken (STAT−/Ken−) showed no activation after pathway stimulation and did not respond to Ken (Arbouzova, 2006).

These results indicate that Ken functions as a transcriptional repressor in this cell-culture system and shows that this effect is specific to the DNA sequence determined by SELEX and EMSA. This result is also consistent with a recent whole-genome RNAi-based screen, which used a reporter containing STAT+/Ken+ binding sites and includes Ken among the list of JAK/STAT regulators identified. In addition, recent reports have also demonstrated BCL6 binding to STAT6 sites in vitro and have shown that BCL6 can act as a repressor of STAT6-dependent target gene expression in cell culture. Although this repression is mediated by the binding to corepressors to the BTB/POZ domain of BCL6, no link between BCL6 and STAT activity has been demonstrated in vivo (Arbouzova, 2006).

Finally, it should also be noted that both the STAT+/Ken+ and STAT+/Ken− reporters contain additional GAAA sequences that are not part of the characterized STAT92E binding sequences. However, despite the presence of these potential Ken binding sites within 15 bp of the STAT92E site, Ken expression did not affect the STAT+/Ken− reporter, suggesting that Ken may require STAT92E to influence gene expression. Although no direct association between Ken and STAT92E has been demonstrated, this possibility cannot be excluded, and further analysis remains to be undertaken (Arbouzova, 2006).

Having established that Ken functions at the level of DNA binding in cell culture, it was asked whether Ken also acts as a transcriptional repressor of JAK/STAT pathway target genes in vivo. For this, the effect of ectopically expressed Ken on the expression of putative JAK/STAT pathway target genes was examined and, given the high levels of maternally loaded STAT92E present at blastoderm stage, focus was placed on targets expressed later in embryogenesis. These include the hindgut-specific expression of vvl, the expression of trachealess (trh) and knirps (kni) in the tracheal placodes, and the dynamic expression of socs36E throughout the embryo (Arbouzova, 2006).

First, the effect of Ken was addressed on trh, whose expression precedes the formation of the tracheal pits in the embryonic segments T2 to A8. Levels of trh are greatly reduced in embryos uniformly misexpressing Ken driven by the daughterless-GAL4 (da-GAL4) line. Many tracheal placodes express little or no trh, and tracheal pits fail to form even in the presence of residual trh. Similar effects are seen in updOS1A mutant embryos lacking all pathway activity. Likewise, downregulation of Kni expression is also observed in embryos misexpressing ken. These results show that both endogenous trh and kni are downregulated by ectopically expressed Ken (Arbouzova, 2006).

Whether Ken can modulate the expression of socs36E, a Drosophila homolog of mouse SOCS-5, was tested. socs36E expression closely mirrors that of upd, showing JAK/STAT pathway-dependent upregulation in segmentally repeated stripes, tracheal pits, and the hindgut. By contrast to trh and kni, ectopically expressed Ken does not affect any aspect of socs36E transcription. However, controls expressing a dominant-negative form of the pathway receptor DomeΔCyt, using the same Gal4 driver line, show a strong downregulation of socs36E, an effect reproduced by the complete removal of all JAK/STAT pathway activity by the updOS1A allele. Taken together, these results illustrate that ectopic expression of Ken during Drosophila development is sufficient to downregulate the expression of only a subset of putative JAK/STAT pathway target genes (Arbouzova, 2006).

As part of this analysis, modulation of vvl by Ken was tested. In wild-type embryos, vvl is expressed in the developing trachea and lateral ectoderm (in a JAK/STAT-independent manner) and in the hindgut of stage 12-14 embryos, where it requires JAK/STAT signaling. In updOS1A mutants, no vvl expression in the hindgut can be detected, indicating that this locus is a target of pathway activation. When Ken is uniformly misexpressed throughout the embryo, vvl expression is no longer detectable in the hindgut. Thus vvl, like trh and kni, can be a target of Ken-mediated repression (Arbouzova, 2006).

Having established that ectopic Ken is sufficient to downregulate vvl in the hindgut, whether endogenous Ken performs a similar role was determined. One overlap between ken expression and regions known to require JAK/STAT signaling are the developing posterior spiracles, structures in which both the pathway ligand upd and ken are simultaneously expressed. However, vvl is never detected in the posterior spiracle primordia in wild-type embryos, despite JAK/STAT pathway activity induced by upd expression in these tissues. Intriguingly, in a heteroallelic combination of the strongest kenk11035 allele and Df(2R)Chig320, vvl transcript was detected not only in its normal expression domain within the hindgut but also in the posterior spiracles. This ectopic expression is initially detected from late stage 13 and rapidly strengthens during stage 14-15. When kenk11035/Df(2R)Chig320 embryos simultaneously mutant for the amorphic updOS1A allele were analyzed, upregulation of vvl in the presumptive posterior spiracles was never observed at the stage by which ectopic vvl expression was first detected in the ken mutant embryos. At later stages, JAK/STAT pathway activity is required for posterior spiracle morphogenesis, posterior spiracles do not form, and upregulated vvl is not present (Arbouzova, 2006).

These results demonstrate that Ken is not only sufficient to downregulate the JAK/STAT pathway-dependent expression of vvl in the hindgut, but its endogenous expression is also necessary for vvl repression in the posterior spiracles. In ken mutants, ectopic vvl expression in the posterior spiracles results from a derepression of endogenous STAT92E activity (Arbouzova, 2006).

The overlap between the consensus sequences bound by STAT92E and Ken, together with the analysis of reporters containing STAT+/Ken+ and STAT+/Ken− binding sites, indicate that Ken is likely to selectively regulate only a subset of JAK/STAT target genes. In this model, some target genes are regulated by binding sites compatible with both STAT92E and Ken, while others contain sequences to which only STAT92E can associate. While the DNA binding site is critical in cell-culture systems, similar proof is more difficult to establish in vivo. In particular, only a limited number of JAK/STAT pathway target genes have been rigorously demonstrated to require STAT92E binding in vivo (Arbouzova, 2006).

Although studied in some detail, the regulatory domains controlling vvl expression in the developing hindgut have not been identified. Therefore, although these results predict that such a domain would contain STAT+/Ken+ binding sequences, further analysis is required to confirm this hypothesis. By contrast, the regulatory domain of socs36E required to drive gene expression in the blastoderm, tracheal pits, and hindgut comprises a 350 bp region containing three STAT+/Ken+ and two STAT+/Ken− binding sites. Although not conclusive, the presence of STAT92E-exclusive sites in this region may explain the inability of Ken to downregulate socs36E in vivo (Arbouzova, 2006).

The findings also draw a parallel between Drosophila Ken and BCL6. The data presented demonstrate that both proteins show similar abilities to bind DNA and to mediate transcriptional repression with some evidence also linking BCL6 to JAK/STAT signaling as described here. Taken together, these similarities suggest that Ken and BCL6 represent functional orthologs of one another. Given this evolutionary conservation, it is tempting to speculate that the selective regulation of JAK/STAT pathway target genes is also conserved and may represent a general mechanism by which the pathway is modulated to elicit diverse developmental roles in vivo. Although many STAT targets undoubtedly remain to be identified, it will be intriguing to see which may also be coregulated by Ken/BCL6-dependent mechanisms (Arbouzova, 2006).

Genome-wide mapping of in
vivo targets of the Drosophila transcription factor Krüppel

Krüppel (Kr), a member of the gap class of Drosophila segmentation
genes, encodes a DNA binding zinc finger-type transcription factor. In addition
to its segmentation function at the blastoderm stage, Krüppel also plays a
critical role in organ formation during later stages of embryogenesis. To
systematically identify in vivo target genes of Krüppel, DNA fragments were
isolated from the Krüppel-associated portion of chromatin and they were
used to find and map Krüppel-dependent cis-acting regulatory sites in the
Drosophila genome. Krüppel binding sites are not enriched in
Krüppel-associated chromatin and the clustering of Krüppel binding
sites, as found in the cis-acting elements of Krüppel-dependent
segmentation genes used for in silico searches of Krüppel target genes, is
not a prerequisite for the in vivo binding of Krüppel to its regulatory
elements. Results obtained with the newly identified target gene(s) ken and
barbie, together referred to as ken indicate that Krüppel
represses transcription and thereby restricts the spatial expression pattern of
ken during blastoderm and gastrulation (Matyash, 2004).

To establish whether the newly identified candidate genes are indeed
regulated in a Krüppel-dependent fashion, focus was placed on ken.
The reason for this choice was that ken, which encodes a DNA binding zinc
finger-type transcription factor, appears at a first glance unlikely to be a
Kr target gene. This is because (1) Kr activity is not required for
male genitalia formation and adult eye development, the two processes in which
ken is involved. (2) ken is expressed early in two stripes
that do not overlap with the Kr expression domain during blastoderm stage
and gastrulation. In contrast, it was found that the isolated 749-bp DNA fragment is highly enriched in the DNA of
Krüppel-associated chromatin and that
it contains five Krüppel binding sites confirmed by gel mobility shift assays (Matyash, 2004).

To solve this apparent dilemma and to thereby demonstrate that the screen has
indeed led to Krüppel target genes, it was asked whether Krüppel does
regulate ken expression in vivo by performing in situ
hybridizations of ken probes to whole mount preparations of wild type and
homozygous Kr1 lack-of-function mutant embryos.
In wild type, Krüppel is
initially expressed in a broad band in the central region of the blastoderm.
In contrast, ken is expressed in two distinct
stripes that are anteriorly adjacent and posterior to the Kr central
domain. In Kr mutant embryos, the two stripes
of ken expression are not altered, but an additional expression domain was observed
where Kr is normally expressed
at syncytial blastoderm stage. This expression domain appears earlier than the normal stripes of
ken expression, and it subsequently fades in a posterior to anterior
direction, resulting in a third narrow stripe that remains separated from the
anterior ken stripe.
These observations establish that in the absence of Kr activity,
ken is activated in the central region of the embryo and that this aspect
of ken activity is normally repressed in a Krüppel-dependent manner (Matyash, 2004).

Previous results have shown that the expression of the anterior stripe of
ken is activated in response to the transcription factors encoded by
bicoid and hunchback, whereas the posterior stripe is activated by
the transcription factor of tailless, and its shape and size are due to
repression by Huckebein. To establish whether ectopic
expression of Krüppel also causes the repression of ken, a
heat shock-driven Kr transgene was used to misexpress
Kr uniformly in the blastoderm embryo.
The posterior stripe of ken expression is
not affected by ectopic Kr activity, whereas the anterior ken
stripe is lacking. Collectively, the results demonstrate that Krüppel
participates in early ken regulation by acting as a local repressor of
the gene in wild type embryos (Matyash, 2004).

This study was directed at the identification of Krüppel-dependent
genes involved in neurogenesis, muscle, and Bolwig organ development. Genes identified that
are involved in neurogenesis include tup, cut and short stop. In fact, 55 of the 82 isolated
genes are known to participate in these developmental processes. Thus, it is expected that
Krüppel regulates possibly several hundreds of genes during the entire
life cycle of the fly (Matyash, 2004).

Two of the Kr target genes (emc and osa) have been
identified in a genetic modifier
screen for gene products that mediate Kr activity. In addition, a DNA fragment
corresponded to the intron of the gene CG7097, a
putative regulatory target of segmentation genes expressed during blastoderm
formation. Microarray-based expression data and whole
mount in situ hybridization of early embryos
shows that this gene as well as additional 29 of the 43 candidate
genes are expressed during the first 14 h
of embryonic development. These observations and the results of the genetic
studies with ken indicate that the DNA isolated from
Krüppel-associated chromatin revealed in vivo target sites of the
transcription factor (Matyash, 2004).

Previous analysis has shown that during segmentation Krüppel controls the
activity of other transcription factors that are part of a cell fate-determining
gene network. The results
suggest that this earlier finding is not restricted to Kr segmentation
function since the majority of the Krüppel target genes identified in this
study (18% of the total isolates) encode transcription factors as well. The more
important notion is, however, that Krüppel not only participates in the
regulation of transcription factor networks at the different levels of the
segmentation gene cascade but also assists signaling
events by regulating various pathway components, as exemplified by target genes
coding for components of the JAK/STAT-signaling pathway. Krüppel target DNA
includes portions of the genes ken, STAT92E, and stc, which code
for JAK/STAT-mediating transcription factors as well as factors known to participate in signaling by the epidermal growth factor receptor (Asteroid)
and Rho GTPases (Gef64C). Moreover, the isolation of genes encoding lipid metabolism-related enzymes and
the lipid carrier Neural Lazarillo (NLaz) suggests
that Krüppel not only takes part in embryonic fat body development
but also participates in metabolic functions (fat storage
or fat consumption) of the organ (Matyash, 2004).

The majority of the newly isolated Krüppel target sites lack Krüppel
binding site clusters as revealed in cis-acting elements of the
Krüppel-dependent segmentation genes.
However, the isolated and subsequently tested set of DNA
fragments is enriched in Krüppel-associated chromatin, as has been found
with the eve stripe 2 element, which contains clustered Krüppel
target sites. This finding suggests that the
clustering of binding sites is not the sole biologically relevant marker for
Krüppel-dependent cis-acting control elements. Furthermore, the algorithm
applied to detect Krüppel binding sites only counted matches of sequences
to a weighted matrix that were arbitrarily set above a certain threshold. In
consequence, functional low affinity binding sites or Krüppel-dependent DNA
segments that contain only few and unclustered high affinity binding sites were
left undetected (Matyash, 2004).

Interestingly, more than half of the Krüppel target DNA fragments (68%)
were located in introns and exon/intron overlap sequences or in exons
and not at the canonical 5' termini of
protein-coding genes. The location of these fragments downstream of the
transcription start sites suggests that they may represent distal regulatory
elements (e.g., enhancers or silencers) or promoters for non-coding RNAs,
as implied by a most recent study on transcription factor binding along human
chromosome 21 and 22. Because noncoding transcripts
within the Drosophila genome are not systematically annotated, it cannot be
decide whether Krüppel participates in the transcription of such
transcripts (Matyash, 2004).

A surprising result of this study was that ken, which is not expressed in
the Krüppel domain of wild type blastoderm embryos, is in fact a target of Krüppel. In the absence of
Kr activity, ken is activated in the central region of the
blastoderm. Thus, in addition to the regulation of ken expression in the
anterior and posterior stripe domains, which involves the activities of
bicoid in cooperation with the gap genes hunchback, tailless, and
huckebein, Krüppel is needed to prevent
ectopic ken activation in the blastoderm embryo. This finding and the
notion that ubiquitous Krüppel expression abolishes ken activity in
the anterior but not in the posterior stripe domain suggest that the two stripes
of ken expression are under the control of separate cis-acting elements,
of which only one mediates repression by Krüppel (Matyash, 2004).

The ken and barbie gene encoding a putative transcription factor with a BTB domain and three zinc finger motifs functions in terminalia development of Drosophila

Mutations in the ken and barbie locus are accompanied by the malformation of terminalia in adult Drosophila. Male and female genitalia often remain inside the body, and the same portions of genitalia and analia are missing in a fraction of homozygous flies. Rotated and/or duplicated terminalia are also observed. Terminalia phenotypes are enhanced by mutations in the gap gene tailless, the homeobox gene caudal, and the decapentaplegic gene that encodes a TGFbeta-like morphogen. The ken and barbie gene encodes a protein with three CCHH-type zinc finger motifs that are conserved in several transcription factors such as Krüppel and BCL-6. All defects in ken and barbie mutants are fully rescued by the expression of a wild-type genomic construct, which establishes the causality between phenotypes and the gene (Lukacsovich, 2003).

The body plan of Drosophila is sexually dimorphic as in the case of the body plans of many other animals. The most obvious sex difference occurs in adult terminalia, i.e., the genitalia and analia. Both the adult genitalia and analia arise from the genital disc, a saclike cluster of primordial cells set aside during mid-embryogenesis for the later development of imaginal terminalia. The embryonic primordium of the genital disc comprises 15­20 cells derived from several adbominal segments posterior to A8 and the nonsegmented telson. Thus, it represents a composite imaginal disc just as the eye-antennal disc. The differences in the genital disc between males and females become apparent near the end of the second-instar larval stage: three primordia, i.e., the female genital primordium, comprising a single genital disc, the male genital primordium, and the anal primordium develop differently depending on sex (Lukacsovich, 2003).

The female genital primordium derived from embryonic A8 grows whereas the male genital primordium does not. In males, the male genital primordium derived from the A9 segment expands, whereas the female counterpart is repressed. The anal primordium consists of cells in A10, possibly in A11 and the telson, and it develops into the imaginal anus in both sexes despite the sexually dimorphic anal structure (Lukacsovich, 2003).

The sex-specific differentiation of the genital disc is under the control of the sex-determination cascade, which involves the Sex lethal (Sxl), transformer (tra), transformer-2 (tra-2) and doublesex (dsx) genes. For example, a clone of cells without the tra gene produced in the genital disc of a female second-instar larva may give rise to both male and female genitalia in adults. Moreover, chromosomally female diplo-X flies bearing the temperature-sensitive tra- 2 mutation, tra-2ts, may develop male genitalia when they experience temperature upshifts during the larval stage or female genitalia when exposed to temperature downshifts at the same stage. The sexual types of the anal plate are also affected: temperature upshift leads to the formation of a male anal plate whereas temperature downshift results in that of a female anal plate (Lukacsovich, 2003).

Pattern formation in the genital disc is accomplished by cell-to-cell communication mediated by diffusible signals such as dpp, wg, and hh gene products, in a manner analogous to that in other imaginal discs. The imaginal structures that are eventually formed by each primordium of the genital disc are different from each other. The fact that the male, female, and anal primordia are the derivatives of different embryonic (para) segments implies that the distinct developmental profile of each primordium is ultimately specified by homeotic gene expression. Indeed, mutations that result in the loss of AbdominalB (AbdB) or spalt (sal) homeotic gene functions transform the tail structures (including genitalia) into those of the more anterior abdominal segments. The homeobox gene caudal (cad) is required for the proper formation of A10 and the telson, including the anus, and is considered to function as a homeotic gene that directs the pathway of anal development. Among the gap genes, tll is known to be involved in terminalia development (Lukacsovich, 2003).

Thus, it is conceivable that morphogenetic cell-to-cell interactions mediated by Dpp, Hh, Wg, and related signals are instructed by homeotic gene products that provide positional cues as well as by the products of sex-determination genes that convey information on which sexual fate to adopt. Although this view provides a general framework for understanding how terminalia differentiate in the genital disc, the molecular mechanism of the system coordinating such complex regulatory networks is poorly understood (Lukacsovich, 2003).

Recent studies revealed, however, that the dachshund (dac) gene is differentially expressed in the male and female genital discs through the sexually different actions of wg and dpp. FGF (fibroblast growth factor) signaling is also crucial for the sexually dimorphic development of the genital disc: FGF expressed only in the ectoderm-derived cells of the male genital disc stimulates FGF-receptorexpressing mesodermal cells to migrate into the male discs. These studies have unraveled some aspects of the complex molecular network underlying the sexually dimorphic development of the genital disc (Lukacsovich, 2003).

In an attempt to elucidate the mechanism of copulation control, a mutant, okina (ok, a name given by Ryu Ueda; Yamamoto, 1997), the homozygotes of which are occasionally devoid of external genitalia and/or analia. The aristae of the mutant flies are unpigmented, thus the name okina, which in Japanese means a respectable old man with a white beard. In subsequent studies, ok was found to be an allele of ken and barbie (ken; Castrillon, 1993), and is referred to as kenok in this study. As inferred from their phenotypic similarity, ken was found to interact synergistically with cad, tll, and dpp. The ken gene encodes a putative transcription factor with three zinc finger motifs, two of which are aligned side by side similar to those found in Kruppel and some other transcription factors. Finally, this study demonstrated that a genomic fragment containing only the ken transcription unit fully rescues all the deficits associated with the ken mutants, establishing the causality between the ken gene and the phenotypes of these mutants (Lukacsovich, 2003).

The kenok allele was isolated during screening for sexual-behavior mutants as well as for those showing low mating success and reduced copulatory duration. ken1 was isolated based on its abnormal genitalia (Castrillon, 1993). Careful examination of external structures revealed that 8% of male flies homozygous for kenok have aberrant terminalia: some of them appear to lack external genitalia, which actually remain inside the abdominal cuticle. On the contrary, there are a few ken mutant flies with duplicated genitalia arranged side by side in a mirror image. There are also flies whose orientation of genitalia is aberrant. Female terminalia is similarly affected by ken mutations at a lower frequency (2%) than male terminalia. Aside from genital anomalies, all ken homozygotes had unpigmented aristae that are somewhat frail physically. Several stronger alleles were identified by genetic complementation tests for reduced viability when placed in trans to kenok or by molecular mapping. These include kenP1244 (l(2)02970), kenP942 (l(2)00628), ken2 (l(2)k11035), and ken3 (l(2)03907). The transallelic combination kenok/kenP942 or kenok/kenP1244 yields flies that display the terminalia phenotype. The terminalia phenotype is 100% penetrant in a few ken1/kenP1244 escapers. The level of expression of the arista phenotype is not increased in these transallelic mutants (Lukacsovich, 2003).

In accordance with the adult genital phenotype, the genital disc was malformed in the ken mutants. There were mutant discs that were split into two subdivisions, which presumably developed into duplicated genitalia as observed in adults. In these experiments, flies carrying a dpp-lacZ reporter construct were used to examine the possible effect of ken mutation on dpp expression. Even in the discs severely distorted by ken mutation, strong dpp expression was observed. In such discs, large changes in the overall disc structure prevented determination of whether the pattern of dpp expression is affected by ken mutation. In accord with the variable phenotypic severity in the ken mutant adults, some mutant discs appear to have a normal structure. dpp -reporter expression in these mutant discs is indistinguishable from that in wildtype discs. This result indicates that either ken mutation does not affect dpp transcription at least in this developmental stage, or the dpp reporter construct lacks ken-responsive sequences present in the genomic dpp gene (Lukacsovich, 2003).

To define the developmental role of ken genetically, the possible genetic interactions of ken alleles with mutations known to affect genital structures were tested. A hypomorphic allele of the homeobox gene cad (cadmd509) markedly increases the frequency of male terminal defects in kenok homozygotes. The cadmd509 allele that carries a GAL4-enhancer trap insertion in the cad locus does not exhibit any discernible phenotypes in external structures including aristae and terminalia on its own, in contrast to cad1, which leads to the loss of anal plates. The proportions of kenok homozygotes with the terminalia defect were 8% in the
cad wild-type background, 30% in the cadmd509 heterozygous
background, and 87% in the cadmd509 homozygous background (Lukacsovich, 2003).

Among the gap genes, tll appears to play an important
role in terminalia formation, as a weak allele (tllle3) is known to lack portions of the anal pads and hindgut. Interestingly, halving the gene
dosage in the tll locus increased the proportion of
kenok homozygous flies with the terminalia defect. Thus, tll and ken appear to function synergistically in genital disc development. Another
locus that found to interact with ken was dpp. The frequency of observing the terminalia phenotype in kenokhomozygotes is increased from 8% to 31% by replacing a wild-type copy of dpp with a mutant allele, dpp (Lukacsovich, 2003).

Thus, kenok mutation created a sensitized genetic
condition, where a small reduction in tll or dpp
activities to levels that does not produce any discernible
phenotypic effect on their own results in explicit abnormality in development. The observed interactions between ken, tll and dpp imply that these
three genes function in the same transduction pathway
or in different signaling pathways that cross talk to each other (Lukacsovich, 2003).

The ken locus has been mapped at 60A in the
right arm of chromosome 2 (Lukacsovich,
1999). Both the arista and terminalia phenotypes of kenok result from the P-element
insertion at 60A because its excision yields revertants. The genomic DNA fragment flanking the
P-element insertion was recovered by the inverse PCR method, and used to identify
recombinant P1 phages with the sequence identical with that flanking the P-element insertion. The
genomic sequences of two P1 phages designated as DS00692 and DS06090 hybridized with the genomic
sequence of the putative ken region. The inserts of the two P1 phages covered the genomic
region of more than 20 kb surrounding the kenok
P-element insertion. The insertion sites of the P-element
in kenok, ken1, kenP942, kenP1244, and ken2 were determined by sequencing. The P-elements in ken2, kenP1244, ken1, and
kenok are inserted between the translation start site
and the transcription start site of the ken gene. kenP942
bears the P-element insertion 375 nucleotides upstream
of the transcription start site (Lukacsovich, 2003).

The conceptual translation of cDNA nucleotide sequence revealed that the open reading frame of
the ken gene can encode a protein of 601 amino
acids. After the submission of the ken
DNA and protein sequences to the GenBank database
(accession numbers: AB010260-61), an identical
sequence was published by Kühnlein (1998). Blast search revealed that the ken protein
has three C2H2-type zinc finger motifs in its C terminus. The region
composed of 47 amino acids from a.a. 500 to 546 contains the first zinc finger motif and part of the
second zinc finger motif. Interestingly, this region
is highly homologous to the zinc finger motifs in humans, rather than to those in Drosophila, Kruppel
and BCL6 proteins. The identities
of this region in ken with those in the human
Kruppel and BCL6 are 55 and 57%, respectively.
The conserved domain analysis database (Pfam)
indicates that the N-terminus of the ken protein
has a putative BTB/POZ damain, which is an evolutionarily
conserved protein-protein interaction domain known to affect the chromatin structure. The alignment of sequences of the BTB domain revealed that the
amino acid residues critical for its function are conserved
among the ken and other BTB proteins.
Thus, ken is a putative transcription factor that has
three zinc finger motifs and a BTB domain (Lukacsovich, 2003).

Developmental Northern blot analysis revealed
that ken transcription is developmentally regulated,
i.e., elevated expression level in embryos
and pupae and much lower expression level in larvae. In adult flies, ken expression is sexually dimorphic, i.e., its expression level is much higher in females than in males.
This high expression level in females likely reflects its accumulation in oocytes because two cDNAs
corresponding to ken have been (BDGP library:
GM12839, GM01621) obtained by the screening of an ovary cDNA library. Thus ken mRNA is maternally transmitted to embryos (Lukacsovich, 2003).

Since the ken gene is required for the normal
development of terminalia and antennae, it may be expressed in the genital and eye-antennal discs.
In situ hybridization experiments were carried out in order to examine the expression pattern
of the ken gene in imaginal discs. In Drosophila,
the genital disc forms the genitalia, analia, and
hindgut of adults. The former two structures are different between the
sexes. The eye-antennal disc develops into an eye,
an antenna, a maximal plap, and a hemilateral
head capsule. In situ hybridization experiments were performed
with a single-strand digoxigenin-labeled DNA probe specific to ken mRNA. ken mRNA was
found to be expressed ubiquitously in both male
and female genital discs and eye-antennal
discs at low levels, but no signal was detected in the brain. The genital disc of an enhancer trap-
ken allele for beta-galactosidase activity, and
the ubiquitous expression of ken at a low
level was confirmed . In males, ken expression is intensified
along the margin of the anterior bulbus. In females, ken is expressed in the posterior
compartment along the anterior-posterior border, with medial expansion in the posteriormost
region. In contrast to its low expression
level in the imaginal discs, a high expression level
of ken was reported in the embryo by Kühnlein (1998). In situ hybridization analysis of
ken expression in the embryo confirmed that result (Lukacsovich, 2003).

The ken transcript is expressed in a distinct
spatiotemporal pattern during embryogenesis. At stage 5 (cellular blastderm), two rather faint
stripes can be detected at positions of 64% (anterior
domain expression; AD) and 17% (posterior domain expression; PD) egg length (EL; 0%EL is the posterior most position). At stage 6 (early gastrulation), these two stripes become more
evident and detectable at the region posterior to
the cephalic furrow (CF) and in the hindgut primordium
(Fig. 8B). AD is lost as gastrulation proceeds
(stage 6­8), while PD remains. At stage 15, AD appears again in the foregut and PD
in the hindgut and anal pad (Lukacsovich, 2003).

To demonstrate unequivocally that mutation in
the cloned gene is responsible for ken phenotypes,
a functional rescue experiment was performed in
which an artificially constructed wild-type transgene
was tested for its ability to restore ken mutant
abnormalities. The integrated genomic DNA construct completely
rescued the ken phenotypes. The viability
of kenok homozygous flies recovered to normal, and
no flies lacking the genitalia or with aberrant pigmentation
of antennae were observed. The genomic
ken+ transgene restored the viability of the
lethal allele kenP942, yielding adult flies homozygous
for kenP942 with a proportion that was expected.
These kenP942 homozygotes have normal
antennae, genitalia and analia, indicating that perfect
rescue is attained by expression of the inserted
genomic ken+ transgene. Furthermore, the kenok phenotypes
in terminalia that resulted from synergistic
interaction with cadmd509, tllI49, or dpp 5 are all
restored by introducing the genomic ken+ transgene
into the mutant flies. Rescue of the kenok phenotypes was also achieved
by insertion of a full-length wild-type cDNA driven
by the heat shock promoter. In this case, the rescue effect was limited such that there were
some kenok-homozygous flies with the transgene
that exhibited a mild antennal phenotype. Although the rescuing ability of hs-ken+ was incomplete,
its effect was apparent without any heat shock treatment, presumably due to 'leaky' expression
of hs-ken+ at 25°C. Because of this limitation,
it was not possible to determine the developmental
stage at which the wild-type-ken function is required
for the normal development of terminal and
antennal structures. Taking all these observations
together, the cloned gene encoding the zinc finger
protein is indeed confirmed to be the ken gene (Lukacsovich, 2003).

A novel Drosophila transcription unit, located in chromosome region 60A, encodes a zinc finger protein that is expressed in distinct spatial and temporal patterns during embryogenesis. Its initial expression occurs in a stripe at the anterior and the posterior trunk boundary, respectively. The two stripes are activated and spatially controlled by gap-gene activities. The P-element of the enhancer trap line l(2)02970 is inserted in the 5'-region of the transcript and causes a ken and barbie (ken) phenotype, associated with malformation of male genital structures. The name refers to the absence of external genitalia in mutants (Kühnlein, 1998).

The reporter gene expression of the P-element enhancer trap line l(2)02970 is defined by two stripes during blastoderm stage. This suggests that it is regulated by the cis-acting elements of a corresponding endogenous gene. The cloning of this putative gene was initiated by plasmid rescue experiments resulting in genomic DNA fragments flanking the P-element integration. They were used to conduct a chromosomal walk and to characterize the nearby transcription unit by cDNA and Northern blot analysis. The coding capacity of the 2970-transcription unit was determined by sequencing of three overlapping cDNAs and corresponding portions of the genomic DNA. The transcript contains three exons and the combined cDNAs add up to 2.8 kb matching nearly the size of the 3.2 kb long embryonic poly(A)+ RNA detected by Northern blot analysis. The 2970-transcript contains a single 1803 bp open reading frame that codes for a putative 601 amino acid protein characterized by three diagnostic C2H2 zinc finger motifs. Two zinc finger motifs are connected by the evolutionarily conserved `H/C-link' motif, the third zinc finger is separated by 17 amino acids from the others. Zinc finger motifs define a distinct class of nucleic acid binding proteins suggesting that the 2970-protein acts as a DNA and/or RNA binding protein (Kühnlein, 1998).

To visualize 2970-transcript expression during embryogenesis in situ hybridizations were performed on whole-mount embryos using antisense 2970-RNA probes. Transcripts were first detected at the beginning of cellular blastoderm (early stage 5) forming two circumferential rings around the embryo which cover three cell rows at 64% (anterior domain; AD) and 17% (posterior domain; PD) of egg length, respectively. At the end of stage 5, the transcripts cover the first and seventh stripe domain of the pair-rule gene fushi tarazumarking parasegment 2 (posterior maxillary and anterior labial segment) and parasegments 14 and 15 (posterior abdominal segment 8 to anterior abdominal segment 10), respectively. The AD fades away during gastrulation (stages 6-8) whereas the PD persists until the end of embryogenesis in the posterior spiracles. 2970-transcript expression is also found at stage 16 in the foregut, the proventriculus, the dorsal pouch, the hindgut and the anal pads. Weak 2970-expression is detectable in the tracheal system and segmentally repeated epidermal stripes (Kühnlein, 1998).

The initial expression domains at blastoderm stage suggest that the 2970-transcript is controlled by maternal and segmentation gene activities. The AD depends on the gene bicoid (bcd), the key component of the anterior organizer system, as shown by its absence in embryos from homozygous bcd females. In embryos from females that contain multiple bcd gene copies the AD is shifted posteriorly to 55% instead of 64% of egg length. The AD is also absent in embryos lacking zygotic gap-gene activity hunchback (hb) indicating that AD expression is mediated by the bcd target gene hb. The PD expression is controlled by the maternal terminal organizer system, mediated by the terminal gap-gene activities tailless (tll) and huckebein (hkb). In tll mutant embryos, the PD is absent. In hkb mutant embryos, the PD extends to the posterior pole. Therefore, PD expression is formally activated by tll activity and repressed by hkb activity as it has been observed for the posterior expression domain of the spalt (sal) gene. However, sal is not involved in PD regulation since sal mutant embryos exhibit a normal PD expression (Kühnlein, 1998).

The P-insertion line l(2)02970 fails to complement the enhancer trap lines ms(2)00331 and l(2)08253 which contain P-element insertions in the same chromosomal position. The three alleles are semilethal in transheterozygous conditions and the corresponding flies develop distally unpigmented, less branched aristae as well as malformation of male genital structures as has been described for homozygous ms(2)00331 flies. The targeted gene locus is referred as ken and barbie (ken). Thus, the three P-insertion lines represent alleles of the ken gene locus. The tight association of the P-insertion l(2)02970, which can be reverted to full viability by P-element jump-out experiments, with the 2970-transcription unitsuggests that the zinc finger protein described here represents a ken gene product. Consistent with this view is the fact that the genitalia disc anlagen is derived from PS 14, a region that expresses the 2970-transcript (Kühnlein, 1998).